Antigen conjugation to TMV.

<p>Purified antigens were conjugated to TMV in a 1:1 molar ratio in an amide-carboxylate reaction using conditions optimized for each specific protein. M: Marker (Precision Plus Dual Color Standard- BioRad). T0: start time (mixture of TMV and antigen), R (1–4): independent reactions, T90-T180: time points (minutes) after reaction start. Successful conjugation is demonstrated by reduction in protein and an increase in conjugate product (>250kDa; arrow, or in the gel stack; arrowhead).</p

Recall IFNγ ELISpot and Granzyme B cellular immune response on D100.

<p><b>A.</b> Splenocytes of twice-vaccinated mice were harvested at Day 100 after intranasal vaccination as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194614#pone.0194614.g007" target="_blank">Fig 7</a>. (<b>A.</b>) Splenocytes from TMV-DOST vaccine groups were tested for Granzyme B secretion after antigen stimulation by the low responder (OmpA) and (<b>B.</b>) high responder (SucB) antigens given with no adjuvant (-), dIC, CpG or diGMP adjuvant. (<b>C</b>.) IFNγ responses after TMV-DOST + diGMP are compared to no stimulation, or PBS vaccinated mice (Norm) groups (2 mice per group). Values for both Granzyme B and IFNγ were adjusted positive spots per 10⁶ cells, and groups that show significant augmentation with adjuvants compared to no adjuvant are shown (** p<0.001 and *p<0.05). Means are presented ± standard deviation, and statistical analysis was done via non-parametric t-test.</p

Pathogen challenge with <i>F</i>. <i>tularensis</i> LVS.

<p>C57BL/6 mice (n = 7–10) were vaccinated 2 times, 28 days apart, with either TMV-DOST with adjuvant or with PBS + adjuvant. Mice were challenged with 10xLD₁₀₀ dose of <i>F</i>. <i>tularensis</i> LVS 21 days after the second vaccine and monitored for weight loss and survival. Mice were euthanized when weight loss exceeded 30%. (<b>A.</b>) Mice were vaccinated with TMV-DOST vaccine + CPG, or left untreated, and survival and body weights were recorded (<b>B.</b>) for 21 days. TMV-DOST was administered with di-GMP or left untreated and survival (<b>C.</b>) and body weights (<b>D.</b>) were recorded for 21 days. Statistical significance was determined via Kaplan-Meier Log-rank Test (GraphPad Prism).</p

Systemic IgA response to TMV-DOST intranasal vaccination.

<p>Systemic serum IgA titers against each specific antigen in the pooled sera of mice immunized twice intranasally with the TMV-conjugated tetravalent vaccine as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194614#pone.0194614.g005" target="_blank">Fig 5</a>.</p

Antigen-specific IgG titers post-vaccine 1 and 2 after TMV-DOST vaccination.

<p>C57BL/6 mice were used in a subcutaneous (n = 4) and intranasal (n = 5) immunization with 20μg each TMV-conjugated tetravalent vaccine with or without (neat) adjuvant on day 1 (vaccine 1) and day 35 (vaccine 2). Sera was collected on day 28 (bleed 2) and day 63 (bleed 4) and tested for total IgG by ELISA on plates coated with 5μg of respective protein and quantified against a standard curve of antigen specific antibody. Post-vaccine 1 bleed 2 (b2) and post-vaccine 2 bleed 4 (b4) data is shown for Dnak (A), OmpA (B), SucB (C), and Tul4 (D). Means are presented ± standard deviation, where statistical significance is p<0.001 (*) compared to vaccination without adjuvant, using a nonparametric t-test.</p

Timeline for immunization, serum and spleen collection.

<p>Vaccinations occurred on days 1 and 35, with serum collection on days 0, 28, 49 and 63. Spleen harvest for IFNγ and Granzyme B analysis occurred on days 65 and 100 post-primary vaccination.</p

IgG isotype profile analysis of TMV-DOST (D, DnaK;O, OmpA; S, SucB; T, Tul4) vaccinated mice.

<p>Bleed 4 sera pooled from mice after the second immunization of 20 μg TMV-conjugated DOST antigen vaccine with (dIC, CpG, MF59 or diGMP) or without (neat) adjuvant was analyzed by ELISA against each antigen-using isotype specific IgG1 and IgG2b secondary antibodies to deduce type of immune response. Isotype profile is depicted as a ratio of the two, where direct addition gives total isotype content. <b>A</b>: Subcutaneous immunization profile. <b>B</b>: Intranasal immunization profile.</p

IFNγ ELISpot assay on splenocytes of mice vaccinated two times with TMV-DOST vaccine.

<p>Splenocytes were isolated from 2 mice per group on D65 after intranasal vaccination and incubated on IFNγ antibody coated plates at a final concentration of 10⁵ cells/well. Whole protein antigen stimulants were added at 20 μg/mL, and plates incubated at 37°C in a 5% CO₂, 99% humidity incubator for ~36h. IFNγ secreting cells were detected and developed using Mouse IFNγELISpot Ready-SET-Go! Kit, developed with AEC, and read on an AID ELISpot plate reader. IFNγ secretion from spleen cells stimulated with DnaK (<b>A</b>.) OmpA (<b>B.</b>) SucB (<b>C.</b>) and Tul4 (<b>D</b>.) are compared to no stimulation (no stim), or PBS vaccinated mice (Norm). Values were adjusted positive IFNγ spots per 10⁶ cells, and groups that show significant augmentation with adjuvants compared to no adjuvant are shown (*p<0.05, **p<0.001). Means are presented ± standard deviation, and p values were calculated by non-parametric t-test. Results are representative of two experiments.</p

Subunit protein conjugation to the surface of TMV.

<p>A. A single TMV virion, consisting of a helical single RNA encapsidated by coat protein. B-D single TMV virion with the subunit antigen of increasingly larger sizes conjugated to the surface of the TMV rod.</p